The present invention relates to electron microscopy, and, more particularly, to a method and apparatus for a confocal scanning transmission electron microscope.
The imaging of samples with high resolution and in three dimensions is of critical importance for many fields, such as biology, materials science and semi-conductor development, among others. One of the major tools in modern biology research is the confocal laser microscope (M. Minsky, “Memoir on Inventing the Confocal Scanning Microscope”, Scanning (USA) 10, 128-138, 1988). The principles and elements of operation include a laser beam focused by an objective lens on a sample at a certain focal plane. The back-scattered light, often from fluorescent markers, is separated from the laser beam by a beam splitter and focused on a pinhole aperture, producing a strong signal in the detector. The key to its depth sensitivity is that light generated not at the focal place is out-of-focus on the pinhole and, consequently, produces much less signal in the detector. In other words, the principal light rays for the focal plane are towards the pinhole aperture and those focused above and below the focal plane are excluded, demonstrating the principle of depth sensitivity of the microscope. Thus, there is a strong difference in light intensity on the detector between light originating from the focal plane and light not originating from the focal plane. The beam is scanned in x and y direction in the focal plane to obtain an image from one plane, then the focus is changed to a new plane and again an image is recorded. This process is repeated, thereby stepping through a series of z values, thus obtaining a three dimensional (3D) image. The image is often deconvoluted with the point spread function (equal to the 3D probe shape) to obtain a sharp 3D image.
The main disadvantage of confocal laser microscopy is that the resolution is not better than a few hundreds of nanometers (nm), due to the wavelength of light. To obtain 3D images with a better resolution several other techniques exist, for example, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, atomic force microscopy (AFM), and electron tomography. But all techniques have their disadvantages. NMR has limited applicability and requires large quantities of a sample in solution, X-ray crystallography requires high-quality crystals with many repeating units of the object of interest, AFM is a surface technique only, electron tomography has many practical difficulties due the required tilt series and has limited resolution.
A few years ago it was proposed to build a confocal electron microscope by N. J. Zaluzec (U.S. Pat. No. 6,548,810), which included an electron source, a scan unit, a lens, a specimen, a second lens, a second scan unit, a pinhole aperture and a detector. The main difference between the Zaluzec '810 invention and the laser confocal microscope is that the detection is not through the objective lens, but that a second lens is employed to project the beam on the pinhole aperture. The reason for this is that the amount of back-scattered electrons with exactly the same energy as the beam is extremely low and, therefore, it is not feasible to use the reversed optical path. The use of a second lens and a second scan unit introduces a major problem, since the scan operation has to be aligned and synchronized between two units with nanometer precision, which is practically almost impossible due to external mechanical vibrations, electromagnetic field, temperature fluctuations, and drift and hysteresis of the microscope.
A partial solution to this problem was found at Oak Ridge National Laboratory, avoiding the pinhole and using an aberration corrected (P. D. Nellist, et al., “Direct Sub-angstrom Imaging of a Crystal Lattice”, Science 305, 1741, 2004) scanning transmission electron microscope (STEM). The use of an aberration corrected STEM provided a sufficiently large beam opening angle to provide depth sensitivity, thus optaining the electron optical variant of the wide field microscope. The depth sensitivity with aberration corrected STEM was demonstrated by locating hafnium atoms in a Si/SiO2/HfO2 advanced device structure with a vertical resolution of approximately 7 nm (K. van Benthem, et al., “Three-Dimensional Imaging of Individual Hafnium Atoms Inside a Semiconductor Device”, Applied Physics Letters 87, 034104-1, 2005). Data on biological samples, i.e., conventional thin sections (osmium stained and epoxy embedded) of mammalian cells, showed a depth resolution of 50 nm (N. de Jonge, et al., “3-Dimensional Aberration Corrected Scanning Transmission Electron Microscopy for Biology”, in “Nanotechnology in Biology and Medicine”, ed. Vo-Dinh, T., 2007 (CRC Press), pp 13.1-13.27). The disadvantage of this method is that the image in one z-plane is mixed with a strong out-of-focus signal from the adjacent planes.
What is needed in the art is a method and an apparatus for a high-resolution three dimensional electron microscope.